The present invention relates to a novel bacterial consortium EBC1000 and a method for remedying biologically recalcitrant toxic chemicals contained in industrial wastewater, waste materials and soils using the bacterial consortium EBC1000. More particularly, the invention relates to a novel bacterial consortium EBC1000 capable of remarkably decomposing a chlorine compound of high concentration and waste acidic or alkaline recalcitrant toxic chemicals difficult to be degraded that are isolated from waste soil and water, the recalcitrant toxic chemicals having a chemical oxygen demand (CODMn) of 20,000 to 100,000 ppm and comprising chemicals, such as isopropyl alcohol (IPA), methylene chloride (MC), dimethylamine (DMA), acrylonitrile (AN), hydrosulfite (SHS), butadiene (BD), sodium alkylaryl naphthalene sulfonate (Tamol-SN), tetra sodium ethylene diamine tetra acetate dihydrate (EDTA), ferrous sulfate heptahydrate (FES), tert-dodecyl mercaptan (TDDM), paramethane hydroperoxide (PMH), N-diethyl hydroxyl amine (DEHA), methanol (MeOH), NaOH and CH3CN.
Environmental contaminations, which are caused by recalcitrant toxic substances, often lead to (i) degradation of food stuffs for humans related to the food chain in the biosphere, (ii) prevalence of infection diseases due to deteriorated immunity, and (iii) spread of chronic diseases and energy exhaustion. Once the ecosphere is radically destroyed, it may take several hundred millions of years for the environment of the ecosphere to recover. degradation of food stuffs for human related to the food chain in the biosphere, prevalence of infectious diseases due to deteriorated immunity, spread of chronic diseases and energy exhaustion. Once the ecosphere is radically destroyed, it may take several hundred millions of years for recovery of the environment of ecosphere.
There has been an attempt to remedy recalcitrant toxic substances contained in the industrial wastewater or noxious waste materials dumped into the sea normally, using physical and chemical methods such as incineration, landfill, chemicals, electrolysis, membrane separation or the like. However, these conventional methods also involve economical and environmental problems. The most efficient and safe method that meets requirements in every aspect of environmentology, sanitation, ecology and economy is the biological treatment method. Many studies have been made on the biological methods, for example, using the natural ecosystem such as silt at an estuary. However, these methods could not be a solution of treating a great mass of industrial waste materials.
It is, therefore, an object of the present invention to provide bioremediation to remedy biologically recalcitrant toxic chemicals contaminating industrial wastewater, waste materials and soils, etc., using a trace of useful specific bacterial consortium that exists in the ecological system.
The present invention is directed to a novel bacterial consortium EBC1000 and a method for remedying biologically recalcitrant toxic chemicals and chlorine compounds contained in industrial wastewater, waste materials, soils, or the like using the bacterial consortium EBC1000. More particularly, the invention is directed to a novel bacterial consortium EBC1000 capable of remarkably degrading chlorine compounds of high concentration and waste acidic or waste alkaline recalcitrant toxic chemicals that are isolated from soil and wastewater having CODMn of 20,000 to 100,000 ppm, such as contained in the medical industrial wastewater to be dumped into the sea, e.g., isopropyl alcohol, methylene chloride, NaOH, Na2SO4, dimethylamine or methanol, or as contained in the petroleum industrial wastewater to be dumped into the sea, e.g., sodium hydrosulfite, butadiene, sodium alkylaryl naphthalene sulfonate, acrylonitrile, tetra sodium ethylene diamine tetra acetate dihydrate, ferrous sulfate heptahydrate, tert-dodecyl mercaptan, paramethane hydroperoxide, N-diethyl hydroxy amine, and CH3CN.
The inventor isolated a novel bacterial consortium EBC1000 (KCTC 0652 BP) having a characteristic of rapidly degrading recalcitrant waste materials of high concentration dumped into the sea as well as chlorine compounds, from the soils and wastewater collected from the Ulsan industrial complex and the petroleum chemical industrial complex in Korea. The bacterial consortium EBC1000 consists of nine strains, each having the ability of degrading recalcitrant waste materials. It was found that the bacterial consortium EBC1000 degrades waste acidic or waste alkaline recalcitrant waste materials having the CODMn of 20,000 to 100,000 ppm contained in the medical and petroleum industrial wastewaters to be dumped into the sea by 80% within 7 days, 90% within 10 days and at least 80 to 90% within 240 hours in an aerobic manner. Examples of the waste acidic or alkaline recalcitrant waste materials contained in the medical industrial wastewater of e.g., pH 3 or 14 include isopropyl alcohol (IPA, 10,000 to 20,000 ppm), methylene chloride (MC, 100 to 7,000 ppm), NaOH (changeable), Na2SO4 (changeable), dimethylamine (DMA, 70 to 2,500 ppm), methanol (MeOH, 27,000 to 54,000 ppm), organic matters and antibiotic residues (40,000 to 100,000 ppm) and Clxe2x88x92(4,000 to 33,000 ppm). Examples of the waste acidic or alkaline recalcitrant waste materials contained in the petroleum industrial wastewater of e.g., pH 1.2, 3.0 or 5.0 include sodium hydrosulfite (SHS, (NaSO2)2, 30 ppm), butadiene (BD,changeable), sodium alkylaryl naphthalene sulfonate (Tamol-SN, 4,000 to 20,000 ppm), acrylonitrile (AN,changeable), tetra sodium ethylene diamine tetra acetate dihydrate (EDTA, 20 to 200 ppm), ferrous sulfate heptahydrate (FES, 60 ppm), tert-dodecyl mercaptan (TDDM, 4,000 ppm), paramethane hydroperoxide (PMH, 400 ppm), N-diethyl hydroxyl amine (DEHA, 200 ppm), and CH3CN (changeable).
Accordingly, the present invention is to provide such a novel bacterial consortium EBC1000 capable of degrading recalcitrant toxic chemicals and chlorine compounds, and a method using the bacterial consortium EBC1000 in remedying biologically recalcitrant toxic chemicals and chlorine compounds contaminating industrial wastewater, waste materials and soils.
Now, a description will be made to isolation, identification and activity of the novel bacterial consortium EBC1000.
1. Isolation of Novel Bacterial Consortium EBC1000
(1) Isolation of 40 Strains from Shaking Culture of the Samples
The soil (1 g) and wastewater (10 ml) from the Ulsan industrial complex were inoculated in a waste acidic and alkaline waste liquid medium (prepared by dilution of K2HPO4 (0.65 g), KH2PO4 (0.17 g), MgSO4 (0.1 g), NaNO3 (0.5 g) and 10% waste acidic and alkaline wastewater containing recalcitrant chemicals in medical and petroleum industrial waste liquid, in 1 liter of deionized water, pH 0 to 14) and then in a chlorine compound liquid medium (prepared by dissolving of K2HPO4 (0.65 g), KH2PO4 (0.17 g), MgSO4 (0.1 g), NaNO3 (0.5 g) and pentachlorophenol (PCP, 50 ppm) in 1 liter of deionized water, pH 7.2), followed by shaking culture at 20 to 30xc2x0 C. for more than 5 days.
1 ml of the shaken culture was collected and inoculated in a waste acidic or waste alkaline solid medium (prepared by adding only 1.5% of agar to the waste acidic or alkaline liquid culture, pH 7.0) and then a chlorine compound solid medium (prepared by adding 20 mg of BTB and 1.5% of agar to the chlorine compound liquid medium), followed by incubation at 20 to 30xc2x0 C. for 3 to 10 days.
The individual pure colonies isolated were further inoculated in the above mentioned cultures and then cultured with shaking, thus isolating 40 useful bacterial strains which have a different colony form formed respectively and the same colony after successive transfer culture.
(2) Isolation of 9 Component Strains After the Bacteria Isolated in Step (1) are cultured in the Medium of an Increased Concentration of the Recalcitrant Wastewater
The bacteria isolated in step (1) were in order and individually inoculated in a minimal medium containing 50% of the crude wastewater and 80% of wastewater containing the recalcitrant chemicals of the medical and petroleum wastewater and then a minimal medium containing 500 ppm of the pentachlorophenol compound and subjected to the same procedures of step (1), thus isolating nine competent strains viable in the wastewater of higher concentration of the recalcitrant chemicals.
The nine bacterial strains, which constitute a single bacterial consortium, named xe2x80x9cEBC1000xe2x80x9d, are designated as EBC100, EBC101, EBC103, EBC104, EBC105, EBC106, EBC107, EBC108 and EBC109.
The novel bacterial consortium EBC1000 was deposited in the Korean Collection for Type Cultures in the Korea Research Institute of Bioscience and Biotechnology as the deposition No. KCTC 0652 BP on Aug. 12, 1999.
2. Optimum Conditions for Growth of Bacterial Consortium EBC1000
The bacterial consortium EBC1000 had the best growth in the Luria-Bertani nutrient medium (containing 10 g of bacto-tryptone, 5 g of bacto-yeast extract and 10 g of NaCl in 1 liter of deionized water) at pH 5 to 8 and 25 to 35xc2x0 C. with shaking at 50 to 100 rpm/min for 24 to 48 hours. The bacterial consortium EBC1000 was also well grown in the successive transfer culture under the same conditions.
3. Identification of Bacterial Consortium EBC1000
The individual strains constituting the bacterial consortium EBC1000, designated as EBC100, EBC101, EBC103, EBC104, EBC105, EBC106, EBC107, EBC108 and EBC109 are a mixture of Gram negative and positive bacteria and have unique shape and size. After culture for 24 hours in the Luria-Bertani medium, EBC100 forms a 1mm-diameter circular colony, EBC101 a large about 2 mm-diameter circular colony, EBC103 a thick about 2 mm-diameter circular colony, EBC104 and EBC105 a small about 0.5 mm-diameter circular colony, EBC106 a thick about 3 mm-diameter colony, EBC107 a about 1.2 mm-diameter brownish colony, EBC108 a about 1 mm-diameter yellowish colony, and EBC109 a about 2-diameter double circle. These nine strains are aerobic and air permeable bacteria. EBC100, EBC101, EBC103, EBC106, EBC107 and EBC108 have high viability in the strong acidic condition of pH 3 to 4 and in the strong alkaline condition of pH 9 to 11. EBC104, EBC105 and EBC109 are slow in growth and EBC100, EBC104, EBC105 and EBC109 show mobility.
For the genera of the individual constituent strains of the bacterial consortium EBC1000 included EBC100, EBC101 and EBC103 are the strains of the genus Klebsiella, EBC105 Providencia, EBC104 and EBC109 Escherichia, EBC106 Bacillus, EBC107 Gram-negative bacteria and EBC108 Gram-positive Bacteria.
The characteristics of the individual strains are presented in Tables 1 and 2.
On the other hand, the individual bacterial strains were analyzed for fatty acid Methyl Esters (FAMEs) by gas chromatography.
For the FAMEs assay, there was used the Hewlett Packard series II Gas Chromatograph model 5890A (Microbial ID. Inc., Delaware, USA) with a 25 cmxc3x970.22 mmxc3x970.33 xcexcm separation column, e.g., a capillary column (HP 19091B-102) fused with methylphenyl silicon.
The gas chromatography was performed under the conditions that the carrier gas is hydrogen, the column head pressure 10 psi, the split ratio 100:1, the split vent 50 ml/min, the septum purge 5 ml/min, the FID hydrogen flux 30 ml/min, the FID nitrogen flux 30 ml/min, the FID air 400 ml/min, the initial temperature 170xc2x0 C., the program rate 5xc2x0 C./min, the final temperature 270xc2x0 C., the FID temperature 300xc2x0 C., the temperature of the inlet port 250xc2x0 C., and the injection volume 2 xcexcl.
The FAME graphs were obtained using a microbial identification system software (Microbial ID, Inc., Delaware, USA). A comparison with a standard calibration mixture (Microbial ID. Inc., Delaware, USA) enabled peak identification, and determination of retention time, peak region and peak percentage.
The assay of the FAMEs for the respective strains of EBC100, 101 and 103 revealed that the intracellular fatty acids composition is C12:0, C14:0, C16:0, C16:1, C17:0 cyclo and C14:0 3OH for EBC100; C12:0, C14:0, C15:0, C16:0, C17:0 cyclo and C14:0 3OH for EBC101; and C14:0, C15:0, C16:0, C17:0 cyclo, and C14:0 3OH for EBC103.
4. Isolation of Constituent Strains of Bacterial Consortium EBC1000
The following procedures are performed for isolating the individual strains from the bacterial consortium EBC1000.
When cultured in the Luria-Bertani agar solid nutrient medium (containing 10 g of bacto-tryptone, 5 g of bacto-yeast extract, 10 g of NaCl and 1.5% agar in 950 ml of deionized water), EBC106 (Bacillus) is characterized by a thick wrinkle peculiar to the Bacillus, EBC107 (Gram negative bacteria) a brownish colony, and EBC108 (Gram positive bacteria) a yellowish colony.
For the other strains the respective colonies can be identified by dilution-smearing the bacterial consortium EBC1000 in desoxycholate agar containing 10 g of bacto-peptone, 10 g of bacto-lactose, 1 g of sodium desoxycholate, 5 g of sodium chloride, 2 g of dipotassium, 1 g of ferrous citrate, 1 g of sodium citrate, 15 g of bacto agar and 0.03 g of neutral red in 1 liter of deionized water (Difco manual, 1984). After cultured for 24 hours in the desoxycholate agar, EBC100, EBC101 and EBC103 have white-in-red spots with viscosity and are slightly different in size, EBC104 white-in-red spots without viscosity, EBC105 brownish spots with viscosity, EBC106 and EBC107 light brownish spots with viscosity, EBC108 colorless spots with viscosity, and EBC109 red spots with viscosity (See. Dictionary of Microbiology and Molecular Biology, 2nd, Paul Singleton Diana Sainsbury, 1987).
5. Characteristics of Bacterial Consortium EBC1000
(1) Decomposition Velocity of sodium Alkylaryl naphthalene sulfonate (Tamol-SN) by Bacterial Consortium EBC1000
Batches of Tamol-SN with an increasing concentration of 500 ppm, 1000 ppm, 2000 ppm and 4000 ppm were added to the minimal medium (pH 7.2) prepared by dissolving 0.065 g of K2HPO4, 0.017 g of KH2PO4, 0.1 g of MgSO4 and 0.5 g of NaNO3 in 1 liter of deionized water. After inoculation of the bacterial consortium EBC1000, the absorbance and the Tamol-SN concentration were measured with an elapse of time. The results are presented in Table 3.
As shown in Table 3, the decomposition velocity per hour of Tamol-SN was 1.3 mg/l/h at the Tamol-SN concentration of 500 ppm and 4.0 mg/l/h at the Tamol-SN concentration of 4000 pm.
(2) Decomposition Velocity of pentachlorophenol Compound by bacterial Consortium EBC1000
Batches of pentachlorophenol compound with an increasing concentration of 200 ppm, 500 ppm, 1000 ppm and 2000 ppm were added to the medium prepared by dissolving 20 mg of BTB in 1 liter of deionized water under the same conditions of the minimal liquid medium as described in (1). After inoculation of the bacterial consortium EBC1000, the absorbance and the pentachlorophenol compound concentration per hour were measured. The results are presented in Table 4.
6. Treatment of Recalcitrant Industrial Wastewater with Bacterial Consortium EBC1000